Glycated proteins, specifically glycated albumin and glycated hemoglobin, are useful indicators of diabetic control. Hemoglobin, albumin and other plasma proteins are continuously glycated in the blood stream, nonenzymatically, by the reaction of glucose with the terminal free amino group of the protein, slowly forming a Schiff base which subsequently undergoes an Amadori rearrangement to form an essentially stable ketoamine.
The degree of glycation of these proteins is directly proportional to the circulating glucose concentration. Thus, measurement of the degree of glycation, specific or nonspecific, provides an accurate reflection of blood glucose levels over the life-span of that protein.
Due to the life-span of hemoglobin, levels of glycated hemoglobin represent an indicator of average blood glucose levels, and therefore diabetic control, over a period of the previous one to two months. Albumin and other plasma proteins have a much shorter serum half-life (approximately 14 days for serum albumin) than hemoglobin. However, because of this fact these proteins serve as useful indicators of average blood glucose levels over the previous 1-3 weeks. Such indicators are of value in unstable diabetics who require careful monitoring or in the evaluation of revised drug, diet or insulin regimens.
A currently accepted methodology for the isolation of glycated hemoglobin, albumin and other proteins is affinity chromatography performed using boronated agarose columns. Assays for both glycated hemoglobin and glycated proteins are disclosed in U.S. Pat. No. 4,269,605 to Dean.
Utilizing currently known techniques, in order to measure for both glycated hemoglobin and glycated albumin in a sample, two separate columns must be run. A whole blood hemolysate is used in the assay of glycated hemoglobin and a serum or plasma sample used to assay glycated albumin. A number of kits are commercially available for separating the glycated hemoglobin from the non-glycated hemoglobin. One such kit is made by Isolab, Inc. and sold under the trademark Glyc-Affin.RTM. GHb. An accessory kit sold under the trademark Glyc-Affin.RTM. PGA is used with the former to measure glycated albumin in serum or plasma.
As previously stated, in order to measure glycated hemoglobin a sample of whole blood hemolysate is used. This hemolysate contains both hemoglobin and plasma proteins, including albumin. However, up to now, colormetric interference from the hemoglobin in the sample was thought to prevent accurate colormetric evaluation of glycated albumin from the same eluate. Additionally, the resulting eluate contained an amount of albumin below the capable detection range of the measuring assays. Turbidimetry refers to the measurement of transmitted light intensity at a 0.degree. angle. Immunoturbidimetry is essentially the same process whereby the turbidity in solution is created by an antigen/antibody complex. This process has been used in the calculation of glycated albumin content from serum or plasma (Reed P, Bhatnagar D, Dhar H., Winocour P. (1986) Precise Measurement of Glycated Serum Albumin by Column Affinity Chromatography and Immunoturbidometry. Clinica chimica Acta 161:191-199). In an analysis for glycated albumin, albumin is the antigen to which the an anti-albumin antibody complexes. This insoluble complex produces a measurable turbidity in the solution proportional to the amount of antigen present and is measured by absorbance. However, as previously stated, this same process was not performed on an eluate of a whole blood sample due to the colormetric interference with hemoglobin. Additionally, it had not been shown that albumin could be separated into its glycated and nonglycated forms in a whole blood sample using current methodology.
Due to the deficiencies in the prior art and, specifically, the inability of known testing methods to isolate and quantitate glycated hemoglobin and glycated albumin from a single affinity chromatography column, the subsequent invention is herein presented.